Biological molecular motors carry out mechanical functions by coupling conformational changes to substeps in chemical fuel consumption. Gyrase is an essential motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. Here we propose a comprehensive biophysical approach for studying gyrase dynamics and mechanochemical coupling. Our goal is to define a set of functionally relevant structural intermediates of the gyrase nucleoprotein complex, and characterize the mechanics and chemistry of transitions between these intermediates. In recent work, we have measured structural dynamics using a single molecule assay in which gyrase drives the processive, stepwise rotation of a submicron fluorescent sphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and simultaneous measurements of DNA contraction revealed multiple ATP-modulated structural transitions. We showed that a critical DNA wrapping step is coordinated with the ATPase cycle and proceeds via an unanticipated structural intermediate that dominates the kinetics of supercoiling. We proposed a mechanochemical model that quantitatively explained our findings, featuring a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction. Our model provides a new framework that guides the investigations in this proposal. In the proposed work, we will exploit new instrumentation and methods that we have recently developed. We can now measure twist and torque in individual stretched DNA molecules with spatiotemporal resolutions that far exceed any previously reported methods, using gold nanospheres as low-drag rotational probes. This technology provides access to millisecond-resolution dynamics of DNA gyrase. We further propose to complement our DNA-centric measurements of nucleoprotein dynamics (relying on observations of changes in twist and extension) with simultaneous measurements of protein domain movements using single molecule fluorescence. Finally, we will use complementary structural approaches to characterize the architectures of substates identified in our single molecule studies. The methods established in this proposal are expected to be an important contribution in themselves, and should be directly applicable to observing the structural dynamics of diverse nucleoprotein complexes ranging from nucleosomes to preinitation complexes.